The major goal of the research proposed is to develop a detailed spectroscopic approach to probe the high spin ferrous active sites present in a large number of mononuclear non-heme iron enzymes involved in dioxygen reactions. These enzymes are generally considered not to be spectroscopically accessible due to their lack of visible absorption and EPR features. The approach to this important problem being pursued here is to use the combination of absorption (Abs), circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopies to probe the d-- greater than d excited states, and the variation with temperature and magnetic field of the MCD spectrum (VTVH MCD) to probe the ground state sublevel splittings. This data is then interpreted using ligand field theory to obtain an experimental estimate of the splitting of the d orbitals which probe the geometric and electronic structure of the ferrous active site. Further, the effects of substrate, cofactor, and small molecule interactions with these ferrous active sites should provide molecular insight into the structural basis for reactivity. Excited stat spectral studies are also proposed for two additional derivatives of the non-heme iron enzymes. The nitrosyl derivative of the ferrous sites will be studied by the combination of Abs, CD, and VTVH MCD alone with resonance Raman spectroscopy to probe for difference in the Fe-NO bonding which correlate with differences in dioxygen activation. Further, functionally important ferric forms of the non-heme iron enzymes will be studied using low temperature MCD to obtain the ligand field transitions which probe the geometric and electronic structure of the oxidized form of the active sites. Systematic chemical and spectroscopic studies are proposed for a series of enzymes which are representative of the important classes of non-heme ferrous enzymes: superoxide dismutase (the prototype of the non-heme iron site for which one can correlate the electronic structure with a known geometric structure); lipoxygenases (fatty acid metabolism and regulation of prostaglandin synthesis); extradiol dioxygenases (tryptopha and tyrosine metabolism); tetrahydropterin dependent hydroxylases (phenylalanine metabolism related to mental disorders); alpha- ketoglutarate dependent dioxygenases (collage synthesis); monooxygenases (hydrocarbon and fatty acid hydroxylation); bleomycin (anticancer drug involved in DNA cleavage). Initial results on SOD and lipoxygenase demonstrate that this excited state spectral approach provides a powerful new probe of the active sites in these non-heme ferrous enzymes.